The objectives of this continuation grant are to characterize the molecular, cellular and biochemical processes of genotoxicity in human kidney and skin epithelial cells. Specifically we will address the hypotheses that DNA repair plays a critical role in protecting human cells from the cytotoxic, mutagenic and carcinogenic effects of environmental agents. Over the past 5 years we have extensively studied the processes responsible for repairing premutagenic/procarcinogenic O6-alkylguanine (O6- alkylGua), O4-alkylthymine (O4-alkylThy) and pyrimidine dimers in cell-free extracts and cell cultures derived from human kidney and skin. These studies have provided new insights into repair activity in tissue and cultured cell populations. With the emergence of molecular and cellular techniques, we are now beginning to focus on the organ, cell and gene specific responses to genotoxin exposure. Two organs, human kidney and skin, of differing activity for repairing O6-alkylGua, will help determine the organ specific responses to genotoxins. Within these 2 tissue and in cells cultured from the tissues, in situ hybridization techniques are beginning to identify, in vivo, the cellular diversity of the O6-methylGua- DNA methyltransferase (O6-MT) mRNA. This is especially important since the O6-MT is consumed during the repair of the O6-alkylGua adduct. Thus this system should establish the organ and cell specific susceptibility to genotoxins. Once the distribution of DNA repair in the various cell types of the kidney and skin tissue is established, the nature and spectra of mutations induced by O6-EthylGua and O4-EthylThy and modified by DNA repair will be determined in these cell types cultured from these organs. The mutational analyses will utilize the well characterized coding sequence of the HPRT gene. These studies will lead to a better understanding of the molecular mechanisms of DNA repair in genotoxicity and its relationship to the organ and cell specific sensitivity to the cytotoxic, mutagenic and carcinogenic effects of alkylating agents. Thus, the studies of this renewal grant application are designed to provide new information and insights into those pivotal questions relating alkylation of DNA and DNA repair processes to human genotoxicity.